This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In vertebrates, hyaluronan (HA) is an extracellular matrix polysaccharide that occurs in a free state in soft connective tissue where it conveys its viscoelastic properties and acts as an intercellular spacefiller. HA also interacts with proteins (via Link module domains) and can form large macromolecular complexes that are important in extracellular matrix assembly and remodelling and provide turgor in cartilage and other tissue. At present, the local solution structure of HA is subject to debate and little is known about the three dimensional organisation of the macromolecular HA-protein complexes thus limiting our understanding of the extracellular matrix and various diseases, e.g., arthritis, cancer, and infertility. Recently, we have shown how atomic-scale simulations of HA fragments (e.g., 8 saccharides long) may be used to help interpret nuclear magnetic resonance (NMR) experiments. Simulations have provided much insight into the local conformational state of hyaluronan in solution which is an important determinant for the viscoelastic and hydrodynamic properties of HA. Similarly, a high-resolution structure for a recombinant HA-binding domain (Link module) from the protein TSG-6 (denoted Link_TSG6) in both its HA-bound and un-liganded forms, has been obtained from NMR data. By exclusive use of NMR it has not been possible to characterize the orientation/conformation of the HA ligand de novo. However, the use of experimental data and structural constrains from molecular modeling have facilitated construction of a preliminary model of the binary complex (1). Expression constructs have been produced for other Link module containing proteins, namely the G1-domains of aggrecan and versican, cartilage link protein and link protein 3 that all contain a contiguous pair of Link modules in their HA-binding domains. These protein domains facilitate the attachment of proteoglycans to HA chains, responsible for the formation of large macromolecular aggregates. The interaction is often mediated through a ternary complex between HA, the proteoglycan and a stabilising 'link protein'leading to a structure that is essentially undissociable. Homology modeling, combined with constraints deduced from the likely HA binding site, has allowed models of the double link modules to be constructed for these proteins (2). RESEARCH FOCUS: Our main goals are (a) characterizing the molecular interactions that occur between HA and the variety of Link module containing proteins and (b) determining the three dimensional organisation of supramolecular HA-protein complexes by using novel compounds such as specifically isotopically enriched hyaluronan and specific HA-binding protein expression constructs together with high-field NMR and X-ray crystallography. However, the HA polymer is dynamic and a full understanding of the molecular interactions between HA and the proteins it binds will necessitate an investigation of molecular dynamics. In this regard, we have already modeled the Link module from TSG-6 using an all-atom approach in the presence of explicit solvent and ions and have shown that there is a significant overlap with experimental NMR data. We aim to use computational modeling to aid the process of understanding dynamic interactions between Link modules and HA and their supramolecular organisation. In particular, we intend to model HA-protein complexes using all-atom molecular modelling and eventually coarse-grained molecular modelling. The large molecular complexes that we are aiming to model necessitate more computational resources than we currently have at our disposal. This PSC project has three Specific Aims: I) Molecular modeling of protein-carbohydrate complexes using restraints from newly obtained NMR data. II) All-atom molecular dynamics simulations of protein-carbohydrate complexes using explicit solvent and ions to provide information about interfacial dynamics and to help interpret experimental data. III) Model interactions between multiple proteins and large molecular mass HA using coarse-grained modeling. Computational Requirements: Molecular mechanics minimization/molecular dynamics of 20-30,000 atoms with CHARMM program on Lemieux, Rachel or Jonas. REFERENCES 1. Blundell, C. D.;Mahoney, D. J.;Almond, A.;DeAngelis, P. L.;Kahmann, J. D.;Teriete, P.;Pickford, A. R.;Campbell, I. D.;Day, A. J., J Biol Chem 2003, 278, 49261-70. 2. Blundell, C. D.;Almond, A.;Mahoney, D. J.;DeAngelis, P. L.;Campbell, I. D.;Day, A. J., J Biol Chem 2005, 280, 18189-201.